help me setup DIY-BIO home Lab to produce Recombinant Factor VIII

[Sunil Phani]

can someone please help me setup DIY-BIO home Lab to perform mammalian cell culture to produce  Recombinant Factor VIII protein with  mammalian cells(CHO, HKE 293, HEL, etc) and kindly provide me recombinant dna protocols for identification, isolation , extraction, insertion and expansion .my requirement is to prepare pilot project in small scale DIY-BIO lab which can further be scaled.

if possible provide me vectors, cell line and seedstock. i am new to DIY-BIO hence i need helping hands of veterans like you ppl .. your help be highly appreciated

[John Griessen]

On 01/16/2015 08:01 PM, Sunil Phani wrote:
> can someone please help me . . .

[huge list, usages like "provide me", "I need", "help me", and no
depth of study demonstrated by statements of a new list member]

is a classic homework request, but yours seems like some kind of
manufacturing business app, so my first question is,

Would you like a profit analysis with that?
Maybe a business plan write up on the side?

But, really, engineering proteins while using human cell cultures is outside the scope of DIYBIO as I understand it.
Even mammalian cell culture has big biosafety impacts as I understand it.

[Dakota Hamill]

http://www.goodrx.com/novoseven-rt

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[Sunil Phani]

no sir, its not for some manufacturing business app,

so i dont need profit analysis or  business plan write up

all i want is to do is Formation of recombinant DNA with a cloning vector to produce Factor VIII protein 

Thanks and regards 

Sunil Phani

[Sunil Phani]

can some one though light on Gibson assembly approach for a cloning a vector to produce Factor VIII protein

will the size of the DNA to be cloned have any constraints with Gibson assembly approach because the size of Coagulation factor VIII is Size: 2351 amino acids; 267009 Da

[Dakota Hamill]

I'm not usually one to try to dissuade people from trying to achieve things others think is impossible, but you should stop and take a step back and look at the scope of your project.

You're trying to make a biologics drug that some company has poured probably hundreds of millions of dollars into, with access to some of the most advanced equipment and best minds in the world, and even at their industrial scale production of this compound, it still costs something like $1.60 per MICROgram. 

If it was as easy as making a plasmid and putting it in E. Coli, it'd have already been done.  I have no idea how it's made but read that they use some weird cell line of a hamster or some other rodent.  Ya, never-mind, the link that I already posted has that information.  There are more steps after it's even excreted by the cells, including some catalyse treatment step and then a separation step.

http://www.rxlist.com/novoseven-drug.htm

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[Sunil Phani]

hey thanks for your suggestion and support .. 

the link u have sent is for coagulation Factor VII(F7) 

and the protean i am looking for is coagulation Factor VIII(F8)

they differs by size significantly..

thanks for suggestions once again and looking forward for further help

 

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[SC]

Hi Sunil,

 

You are suggesting a very large project.  What are your skills currently?  Have you cultured mammalian cells?  If you are a beginner with such things, the first thing you should learn is sterile technique.  Even in very well equipped labs, culturing mammalian cells without contamination is challenging.

 

I wish you a lot of luck in your project, but please be patient because the learning curve may be huge.

Stacy

[Mega [Andreas Stuermer]]

It may be smart to take an internediary step? Like gfp in bacteria, then gfp in plant, and then factor VIII in plants or yeast?

[Sunil Phani]

any one got gfp seed stock ??

On Saturday, January 17, 2015 at 7:31:14 AM UTC+5:30, Sunil Phani wrote:

[Mega [Andreas Stuermer]]

You mean a plasmid? For bacteria or plant?

[Sunil Phani]

For bacteria 

On Fri, Jan 23, 2015 at 3:34 AM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

You mean a plasmid? For bacteria or plant?

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[Mega [Andreas Stuermer]]

I only have pVIB. If you want I can send you.

Or you order the GFP plasmid from http://www.carolina.com/biotechnology-plasmid-lambda-dna/pgreen-plasmid-1-ug-200-ul-0005-ugul/211449.pr?catId=&mCat=&sCat=&ssCat=&question=plasmid

With it the colonies are even green under normal light

[Mega [Andreas Stuermer]]

Alright, I send you the DNA dried on filter paper. All you have to do is elute it in te buffer or sterile desionized h2o in a 1.5 mL tube. Do you have the equipment to handle?
It would probably make very much sense to ask the next university if you can do the experiment in their lab.

[Filip Hasecke]

Hi Sunil,

sorry to be the fun killer, but you should forget about home producing clinical-grade protein therapeutics. Slight contaminations or aggregation of your protein might lead to immunogenic reactions towards your therapeutic. And a second administration of your protein, after the patient developed anti-therapeutic antibodies, might be lethal in the worst case.

In our lab we also work on mice and drug administration of protein therapeutics. Hence I know about the high standards that need to be fulfilled to even keep your mouse alive.. ^^

Those things aside. Unless you are a really rich person, the costs of the equipment that is required for the expression, purification, packaging and storage steps is incredible. You would need a professional clean room to prepare your samples, pharmaceutical grade chromatography equipment, viral filtration, just to name a few...

I like your ambition as a DIY biologist, but the preparation of clinical/pharmaceutical therapeutics is far beyond the scope of a DIY project.

Cheers

Filip

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[Mega [Andreas Stuermer]]

Everyone needs a goal. Target the stars, if you fall you'll land on the moon still. Or so they say.

You can learn how to produce the factor, but on the way you'll learn about immune reactions as well probably. And then you would be able to produce it, but won't want to do it in a basement anymore. But they you can do it in an commercial setting too

[Mega [Andreas Stuermer]]

[Mega [Andreas Stuermer]]

[Sunil Phani]

i welcome your suggestion Filip ... the idea is not to produce the protein to administer to human beings the idea is to go through the entire cycle of recombinent protein production.. just decode the mistress of mammalian cell culture.. basically my philosophy in life is "the person how knows exact direction to his destination can never fork or hack the system" ... i know i am lost but in the detour i may find  some thing which wouldn't have notice in actual thought-up path...

but Filip i really understand your concerns and i will be looking forward for future advices  too fro you 

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[Mega [Andreas Stuermer]]

While the pVIB plasmid is shipping, you might want to look into immunology a bit (though it is a very big and complex field), what would happen if you injected crudely purified protein into a human. What are the negative effects, and how are they induced.

[Sunil Phani]

sure mega .. i'll go through immunology as you have suggested ...

and thanks for that shipment ...

On Mon, Jan 26, 2015 at 3:43 PM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

While the pVIB plasmid is shipping, you might want to look into immunology a bit (though it is a very big and complex field), what would happen if you injected crudely purified protein into a human. What are the negative effects, and how are they induced.

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[Sunil Phani]

guys sorry for the gap completely busy reading some stuff about immunology 

thanks to mega once again for enlisting me deep into immunology , read basics from the following books just loved these books and lot more from internet....

Immunology  by Thomas J. Kindt

Fundamental Immunology by William E. Paul

coming back recombinant protein culture guy please through some light on pros and cons for choosing between Pichia pastoris vs eukaryotes for recombinant protein expression, in this juncture of my aim to produce lab scale prototype of producing recombenant Factor VIII

[Cory Geesaman]

I find is scarier that he wants to culture mammalian cells.  There are a lot of drugs that are over-priced (take for example TA-65 - something like $600 for a 3 month supply last I checked - from about $4 of raw material and maybe $10 of electricity (aside from equipment costs - maybe $100 worth of disposables to make a few years' worth of the stuff - which aren't outside the realm of DIYers).  My experience with the bio side is fairly limited but from what I've gathered thus far if you have to ask about how to culture mammalian cells you shouldn't be.

[SC]

Hi Sunil.  I admire your goal, but your questions are something akin to "How can I perform surgery? And can someone send me a scalpel?"

There are lots of very basic skills you have to master before you can jump into a complex project like this.   For example, working with a sterile cell culture, making recombinant anything (and you shouldn't start with eukaryotes for this, really), and protein purification.  Start slow. Learn the basics.  And read up on immunology.  Also, these procedures can be expensive, especially growing mammalian cells; you can't do it with a light bulb and a shoebox that one might get away with for bacterial cells.   You need to have an understanding of the equipment and supplies you may need, and a realistic budget.

I truly wish you all the best for your project.

[Nathan McCorkle]

On Fri, Jan 30, 2015 at 10:41 AM, 'SC' via DIYbio
<diy...@googlegroups.com> wrote:
> Also, these procedures can be
> expensive, especially growing mammalian cells; you can't do it with a light
> bulb and a shoebox that one might get away with for bacterial cells.

Well I'd argue that you can grow mammalian cells on-the-cheap, but
you've got to be a real expert at this stage because no one has laid
out an instructables or kit-type protocol for this.

Personally I've been wanting to grow some pig (porcine) heart cells
using locally-sourced reagents (i.e. spice shops, grocery stores, and
pharmacies)... and I think it isn't too outlandish... but definitely
time consuming to source a media mixture from mixed 'off-brand'
sources and also rig up lab equipment (petris, reagents, centrifuge
for grabbing plasma from blood, keeping things everything sterile and
clean) at a slaughterhouse/farm-barn. DIY-able, sure, but not as a
beginner unless you're a savant or genius or insanely
dedicated/driven.

[John Griessen]

On 01/30/2015 01:54 PM, Nathan McCorkle wrote:
> definitely
> time consuming to source a media mixture from mixed 'off-brand'
> sources and also rig up lab equipment (petris, reagents, centrifuge
> for grabbing plasma from blood, keeping things everything sterile and
> clean) at a slaughterhouse/farm-barn.

We used to have a local business called Austin Biological Labs that made
culture media, diagnostic test kits, and I worked with them a little
as the FDA was starting to put big test/verify requirements on mfrs like them.
So, I got to see production and testing: steam autoclaves custom made
for production running and clean room. It was a lot of infrastructure
needed to be viable.

They still went under when one main person died. No one left could figure
where $10K/month was going in 1990, so they did a bankruptcy.

[Sunil Phani]

hello friends, 

 i respect your gathered concerns and there is nothing to get alarmed about mammalian cell culture until it's being used up on human beings ...

    and i thoroughly understand the intention behind cautioning me.. i am actually feeling very encouraged and  will be continue to pursue my passion ....

    now i am actually looking into adaptable  approaches  to produce Recombinant Factor VIII using pichia pastoris is a species of methylotrophic yeast. 

    Pichia is widely used for protein expression using recombinant DNA techniques.

    i sincerely believe that you ppl and Diybio will continuously advise me  in this regard and guide me to accomplish my goal rather than plotting the scopes of Diybio 

    "If your actions inspire others to dream more, learn more, do more and become more, you are a leader...."

  

   and in my view Diybio is a leader and savant ppl like you made this group a leader in all aspects ...

   friends pls through some light on pichia pastoris and its pros and cons 

[John Griessen]

On 01/31/2015 08:08 AM, Sunil Phani wrote:
> nothing to get alarmed about mammalian cell culture until it's being used up on human beings ...

It's just that "it" could be used on YOU accidentally as you do lab technique...
and then consider side effects of disease spread risk from your lab cultures, etc.

So, "study up" is all they are saying.

I don't think anyone was alarmed. But...

They care about you even though they don't know you.

They worry when you say non-chalantly, "nothing to get alarmed about mammalian cell culture"...

They worry comparing your non-spell checked emails to all the long sequences of exacting detail
needed to do mammalian cell culture, and worry that "It might not work out..." as Bob Allen,
a good horse trainer says fairly often to his students...

So, "study up", OK?

[leaking pen]

there is nothing to get alarmed about mammalian cell culture until it's being used up on human beings ...

No, that TOTALLY doesn't sound like the beginnings of a Bio-weapon lab. 

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[Sunil Phani]

Pichia pastoris is a species of methylotrophic yeast... "study up", OK? 

[SC]

>They care about you even though they don't know you.

Yep.

 

Also, I'd rather check in here and see a series of small successes from DIYBio-ers than a big plan and no followup.   Sadly, I usually see the latter, and I interpet that as someone giving up.  I could be wrong though.

[Sunil Phani]

guys help me  to choose between CHO or Pichia pastoris or AAV plasmid to synthesize Recombinant Factor VIII on the merits of efficiency ,time and money ,

because to express polynucleotide sequence split into heavy and light chains of Factor VIII in Pichia pastoris expression system is just workaround and will defiantly consume lot of time and obtaining identical sequence for a large protein  as long  as ~220kDa, 2,351 amino acid is not going to be easy  ... where as mammalian expression systems is used extensively till date either with CHO or HEK hence we dont need much of the rework and don't consider mammalian culture as taboo coz its just in vivo culture,,

expecting u guys valued opinion soon /....... 

Regsrds 

Sunil Phani

consults...@gmail.c0m 

[Nathan McCorkle]

On Feb 7, 2015 11:28 AM, "Sunil Phani" <consults...@gmail.com> wrote:
>
> guys help me  to choose between CHO or Pichia pastoris

Well CHO you're gonna need serum and antibiotics (likely) and a CO2 incubator, with Pichia you'll need a supply of methanol. I can't remember details of growing Pichia but it seems like if you can get a culture, it could be easier to grow.... But whether it will express correctly compared to CHO is hard to say.

I take it you've seen this:
http://www.ncbi.nlm.nih.gov/pubmed/16960007

[SC]

Hi Sunil,

It may be helpful to make a list of skills you will need to conduct such a project, such as sterile cell culture,
making a recombinant construct, transfection, protein purification, etc...   Also, a list of equipment and supplies
you will need, along with sources/prices.  This is a normal part of any new laboratory process.

[Jay Hanson]

Hi Sunil

Here is a link to about 30 patents on creating recombinate Factor VIII proteins

If you read these, you will get a feeling for the scope of your project.

http://www.freepatentsonline.com/result.html?sort=relevance&srch=top&query_txt=recombinant+factor+viii&submit=&patents=on

Best,

-jay

[Sunil Phani]

i may be sounding obnoxious but why can we hack a typical UV & RO (ultra violet and reverse osmosis ) water purifier off the shelf

to achieve protein purification ... coz every high end water purifiers now a days promises to be having chromatography and filtration 

i wonder if there is already a DIYBIO chromatography using  off the shelf  UV & RO water purifier coz 

every other UV & RO water purifier discusses deeply how it separates organic and inorganic 

solvents and compounds from water.. then just as appetite to brain why cant we give it a 

thought on its merits 

pros 

• every water purifier have chromatography and filtration mechanism 
•  
• every water purifier have pH, UV sensors, but i don't know weather they have conductivity sensors are not  
• 
  
• every water purifier have filtration mechanism ( may not be 2 µm) 
•  
• every water purifier have say they have ultrafiltration (UF)
• 
  
• most of the UV & RO water purifier have column chromatography (suitable resin is arguable)
• 
  
• most of the UV & RO water purifier have ion exchange chromatography (but we need to  hack it, like we were hacking Hp inkjet printers to print 3d microbial cells  )
• 
  
• needs some reverse engineering to achieve clarified lysate purification  
• 
  
• needs some reverse engineering to achieve HIC by adding ammonium sulphate etc, in recursively cycles 
• 
  
• most of the UV & RO water purifier have TFF but we need to hack  permeate and retentate chambers and focus in recycling renantate several time through diafiltration for efficiency

guys this needs some kind of hacking to off the shelf UV & RO water purifier pls give it a thought and come up with your ideas and viewpoints on this topic., 

because how often can every DIYBIO labs can afford such whopping expensive proprietary chromatographys lets rip open an opensource platform for purifying

proteins  using off the shelf purifiers 

        "No problem can withstand the assault of sustained thinking. ~Voltaire"

[Nathan McCorkle]

On Tue, Feb 10, 2015 at 12:01 PM, Sunil Phani
<consults...@gmail.com> wrote:
> i may be sounding obnoxious but why can we hack a typical UV & RO (ultra
> violet and reverse osmosis ) water purifier off the shelf
> to achieve protein purification ... coz every high end water purifiers now a
> days promises to be having chromatography and filtration
> i wonder if there is already a DIYBIO chromatography using off the shelf
> UV & RO water purifier coz
> every other UV & RO water purifier discusses deeply how it separates organic
> and inorganic
> solvents and compounds from water.. then just as appetite to brain why cant
> we give it a
> thought on its merits
>
> pros
>
> every water purifier have chromatography and filtration mechanism

Generally the goal of water filters is to retain as many molecules of
non-water as possible, and to let water pass.

>
> every water purifier have pH, UV sensors, but i don't know weather they have
> conductivity sensors are

If it is a UV water filter, the UV sensor probably is looking at very
high signal-strengths, and even if the sensor is suitable, the
amplifier/analog-to-digital circuit is probably not sensitive enough
or in the right amplification range. UV sterilization lights will be
VERY bright relative to the amount of light absorbed by presence of an
analyte in solution.

>
> every water purifier have filtration mechanism ( may not be 2 µm)
>

> most of the UV & RO water purifier have column chromatography (suitable
> resin is arguable)

yes the resin in filters would probably be targeting non-polar
molecules, as water is polar... otherwise the resin may simply be
adding a size-exclusion filtration.

> most of the UV & RO water purifier have ion exchange chromatography (but we
> need to hack it, like we were hacking Hp inkjet printers to print 3d
> microbial cells )

It really depends on the type of filter... I bet there are some
filters that allow ions through no-problem, but are targeting things
like viruses, bacteria, pesticides, environmental toxins.

> needs some reverse engineering to achieve clarified lysate purification
>
> needs some reverse engineering to achieve HIC by adding ammonium sulphate
> etc, in recursively cycles
>
> most of the UV & RO water purifier have TFF but we need to hack permeate
> and retentate chambers and focus in recycling renantate several time through
> diafiltration for efficiency
>
>
> guys this needs some kind of hacking to off the shelf UV & RO water purifier
> pls give it a thought and come up with your ideas and viewpoints on this
> topic.,

> becausehow often can every DIYBIO labs can afford such whopping expensive

> proprietary chromatographys lets rip open an opensource platform for
> purifying
> proteins using off the shelf purifiers

Paper is a commonly available chromatography media, agarose and
gelatin are also available.

From a quick google, I get: "Some of the first ion exchangers used
were inorganic and made from aluminosilicates (zeolites). Although
aluminosilicates are not widely used as ion exchange resins used. "
from:
http://chemwiki.ucdavis.edu/Analytical_Chemistry/Instrumental_Analysis/Chromatography/Chromatographic_Columns

[SC]

Hi Sunil,

It's always good to think of new ways to do things.

In this case though, remember that by "protein purification" what you describe is purification of a mixture of all the proteins in the culture.  But what you'd really need to do is purify a single protein and separate it from all other non-target proteins. 

Different types of proteins have different purification methods.  The more similar the target protein is to the non-targets, the more difficult the separation.  You may want to check into chromatography or epitope fusions.

Stacy

[Sunil Phani]

guys sorry for delay in my postings while i was

studying a lot about adeno associated viruses and retroviruses and retroviruses, 

exploring several possibilities guys pls don't be so conventional and conservative 

ofcourse they are safe ...  just think of ex-vivo

why is world of biotech not moving for the past 30 years... 

while other branches of sciences  like Computers and IT emerged in a strong way in 30 years 

we are failed to produce non of the path breaking new ideas at all..

may be too much conservative thinking emerged now a days 

or may be too much churches influence 

or may non presence of openness and democracy in biotech 

[Dakota Hamill]

It has nothing to do with anything you just said.  It has to do with the fact that to make $100 million in IT and computers, you need a laptop and a coffee shop wifi and you can code the new Candy Crush or Flappy Bird and rake in micro-transactions and ad revenue while creating nothing of value besides an addictive phone app and an increase in car insurance premiums.  I like to think of the movie Idiocracy as a good measure of where we're heading.  

To make advances in the life sciences, you need lab equipment that can run into the millions of dollars, as well as the space and facilities to handle the materials you're working with.  While people can get frustrated with the pharma industry, I'm pretty appreciative of the fact that someone can't make my Recombinant Factor VII in a DIY lab and sell it to me.

Be adventurous, dare to dream, but also be a realist.  Everyone on this list is here specifically because we dance around the fringes and may not have found a spot in the conventional biotech industry or academia, but that doesn't mean we don't care about good science, or practical applications.  

I want a spaceship.  Do I blame the church because there hasn't been progression in the field of at home space-ship building?  No.  Because I don't want my spaceship to be built in a garage, I want it to be built by a billion dollar corporation with the best engineers and scientists in the world.

Do I blame the church because I can't make Recombinant Factor VII in my basement and sell it to my friends?  No.  Because I don't want my medicine to be built in a basement, I want it to be built by a billion dollar corporation with the best biologists and chemists in the world.

Does that mean they don't make mistakes?  No.  But I bet their incentives to not make mistakes are greater than Joe Schmoe.

In the end, I have no idea what you even want to do.

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[Nathan McCorkle]

On Sat, Feb 14, 2015 at 9:53 PM, Dakota Hamill <dko...@gmail.com> wrote:

It has nothing to do with anything you just said.  It has to do with the fact that to make $100 million in IT and computers, you need a laptop and a coffee shop wifi and you can code the new Candy Crush or Flappy Bird and rake in micro-transactions and ad revenue while creating nothing of value besides an addictive phone app and an increase in car insurance premiums.  I like to think of the movie Idiocracy as a good measure of where we're heading.  

To make advances in the life sciences, you need lab equipment that can run into the millions of dollars, as well as the space and facilities to handle the materials you're working with.

I've been working at a CPU company for a while now and let me tell you, there's a ton of $ that has to go into R&D as well as production. A big difference I think is that much of biotech is built on instrumentation, so it was (is) in a way limited by the electronics industry. It's only been in the past 10-20 years that compute power, storage, and RAM got to be cheap enough for bioinformatics to take off.  I bet there's also the moral aspect, the public still doesn't 'just know' that biology is not unlike a machine... this concept isn't ingrained in childrens books and cartoons, pop culture. It is increasing, for sure. But when was the last time you heard anything about anti-electronics groups that were on the same level as anti biotech/synbio?

There's been a ton of crazy awesome biotech advancement in the past 30 years, while in computers it's kind of just been "Moore's law as usual"... fun and awesome, but maybe a bit more glamorized and even predictable in some ways.

[Sunil Phani]

 "lab equipment that can run into the millions of dollars"

  that's what it used to be in 80's before home pc's, every thing was under control of big corporates like IBM

  and there used to be people at that time used think that 

 "I want it to be built by a billion dollar corporation with the best" ... in the world

   want run every big banking applications on mainframes built by them then came a revolution like home pc..

  people thought what would we do with a home personal computer .. then there came a revolution like Microsofts licensed

  os for any pc vendor people thought who will care only hardware matters what is in software whay do we even need it..

  then came internet and networking people thought email as a application only geeked university professors then came

  wave of opensource era every thing got liberated and democratized.. 

 "I don't want my spaceship to be built in a garage,

  for god sake you please understand that rockets are being built by may enthusiastics in garages for several years 

  i too can quote movies like October Sky that to its a movie based on true story.

  I blame the church because I can't do research in  Recombinant DNA technology, 

  i cant do research in genetic engineering, gene therapy, embryonic steam cells , Neutrinos, on and on 

 "I want it to be built by a billion dollar corporation with the best    engineers and scientists in the world."  

  do you really think big corporations want to invest there time and money to cure Hemophilia or any disease when they can build a medicine which is needed repeatedly three      times a weak prophylactically earnigs every year with just a single invention some 30 years ago.. do you really think they will venture into gene therapy where they can actually  cure disease with a single  transjugular infusion of AAV to produce missing protein.. i am not trying to sell any thing to any one i am starving to help the children with hemophilia  in improving the quality of life without disability and free of pain by thinking of providing them total care and cure apart from just a temporary fixings which are earning a lot of  wealth to billion dollar corporations and insurance firms...

 and all this frustration and blame not just came coz of your post its because of those universities funded by churches and governments  which don't allow researchers like me to either do reasearch in my intreasted topics and governments which keep putting hurdles to give licences to setup labs and people inside industry thinking every thing should be regulated and moderated  

[SC]

Sunil,

 

Calm down.  

 

Labs that make drugs for sale to people are regulated and licensed because it was quite horrible when they weren't.  

 

I have heard many people claim that Big Pharma has a vested interest in keeping people sick because it is more profitable for them.  I don't believe this any more than I think that the fire department wants you to have a fire hazard in your house or the animal shelter wants to you mistreat your dog.  I have seen more fraud in Little Pharma which skirts regulations than in Big Pharma which is checked and double-checked constantly.   Also, please keep in mind that "Big Pharma" is not one company.  It is a number of companies which are in very keen competition with one another.  If one of those companies found a cure for something, they would rush to get it on the market before one of the other companies did, in a flurry of lawyers and patents.

 

Also, there has been a *ton* of advancements in the last 30 years in biotechnology.  It's a wonderful time to be in this field because there are now discoveries and techniques coming out all the time.

 

Moving on:

 

If you would like to continue your project, I suggest again that you find out which methods you need to use, and what equipment you need to have and how much it will cost.   (I have also been emailing Sunil privately suggesting equipment and supplies for cell culture.)   Also look into how protein purification and how it is done.  It seems you're trying to do a complex project with human proteins and mammalian cell culture on the cheap, when your biggest problem is going to be contamination.

 

I also privately suggested to Sunil that rather than setting up a whole culture lab he may consider having the culture cloned by contract.  I know that would take the DIY out of DIYbio, but it doesn't seem that he is latching on to the DIY bio thing but may be more results-orientied, which is fine.

 

So, Sunil, what equipment and supplies will you need to conduct your project?   (You can start with the list I sent you).  What type of skills are needed (mammalian cell culture, protein purification, making and sterilizing media, etc.)

[John Griessen]

On 02/14/2015 11:33 PM, Sunil Phani wrote:
> while other branches of sciences like Computers and IT emerged in a strong way in 30 years
> we are failed to produce non of the path breaking new ideas at all..
>

Oh, read the instructions for the Dunham chemostats for culturing and it's a lot of work.
Effort levels like that, with long time sequences of exacting steps make for
much more difficult outcomes.

and what Dakota and Nathan said...

On 02/15/2015 02:19 AM, Nathan McCorkle wrote:> it was (is) in a way limited by the electronics industry.

Yep.

It's easier to develop instrumentation parts wise now and with crowdfunding, but still needs a little evolution time
to make bio experiments needing exacting process flows easy and simple to build further on top of.

Your idea that "It should all be now!" and "I want it to be built" miss that point. Everything
moves along at the pace of
1. Ordinary family life first, including baby production, housing affording, education for children.
2. Business steady or booming, not during an economic bust.
3. Patents limiting some areas' opportunity levels, all else considered a go.

So, there's no conspiracy...

Read these instructions and think of how equipment like that could help and
put ideas to the list without blaming anything and you might get some idea generation going.

Your image is not helped by the bad grammar of your posts -- bad grammar/spelling seems lazy compared to
the effort levels needed to get results in science. And your bad grammar is going out to some
of those results-getters, so it may not sound all that great... Perception and response may depend on it some.

[Jeswin]

On Sun, Feb 15, 2015 at 3:47 AM, Sunil Phani
<consults...@gmail.com> wrote:
> I blame the church because I can't do research in Recombinant DNA
> technology,
> i cant do research in genetic engineering, gene therapy, embryonic steam
> cells , Neutrinos, on and on

> and all this frustration and blame not just came coz of your post its
> because of those universities funded by churches and governments which
> don't allow researchers like me to either do reasearch in my intreasted
> topics and governments which keep putting hurdles to give licences to setup
> labs and people inside industry thinking every thing should be regulated and
> moderated
>

I haven't jumped in this thread because I understand that there is a
level of frustration, especially if you live where I think you live
(South Asia?). However, your comments on computer tech progress, big
pharma, and religion/government definitely need citations, not
unqualified generalized perceptions. The realities are much more
complex than people understand. The computer industry is not like the
biotech industry. The former has usefulness to almost 100% of the
population, however the latter is limited to researchers and
hobbyists. Not everyone needs easy access to bio labs so the equipment
is priced by low demand. Increases in demand, such as genomic data by
doctors, lowers the price of gene sequencing. Demand for alternative
fuels will drive down prices in that sector. And so on and so on.
Regarding regulation and ethical considerations, it is necessary even
though it does make things more difficult. There is a reason that
people are wary of using the human experimentation data from World War
II.

All the best in your work.

--
In necessariis unitas, in dubiis libertas, in omnibus caritas.
-Marco Antonio Dominis

[Sunil Phani]

The traditional methods of cutting and pasting DNA seem to be rapidly heading in the direction of "making recombinant DNA by synthesis".

However, for the synthesis route to be adopted by the scientific community, it is important that the mutation rates are minimized, synthesis steps made parallel, technology miniaturised and oligo waste eliminated.

This calls for developing radical new methods of gene synthesis. It appears that in future, bio-CAD (biological computer aided design) tools will be employed to design DNA sequence models and the text file will be emailed to companies for rapid chemical synthesis of recombinant plasmids and microbial genomes. by the way AUTODESK IS DEVELOPING CAD SOFTWARE TO DESIGN, 3-D PRINT LIVING TISSUE its the time we venture into developing  open platform for both hardware and software for biocad 

. 

[Sunil Phani]

yes i need to give you citations phillyj i definitely need to give you citations and references for everything happening around the world  in particularly in pharma development coz some people like you act ignorant and behave as if nothing is wrong is happening and may it be phillyj or some people in this group will never acknowledge children dying with hemophilia or ALD as deeply as my typos...  

because you will always say using mammalian cells or gene therapy illegitimate illogic to use for some conservative thinkers they only preach safety and verbal skills that is nothing but elitist mindset which deviates entire debate by pointing out typos  , 

remember that there are a lot of crazy people in the world 100% against anything that has to do with culturing mammalian cell or manipulating genetics. Going up the route of treating somatic (non-inheritable genes) that are killing little kids is unfortunately the most bullet proof, emotionally knee jerk reaction fueled protection for that uphill battle. Remember that people are still 100% against allowing women access to abortions even in the cases where the mother wanted to carry it to term, but there are medical complications that are going to kill her and the fetus. There are also people against it in the instances of incest/rape. I keep that in mind whenever treading into new turf when it comes to science. It's not the fact that it's difficult to get funding, it's the fact that it's difficult in many cases to tip toe around the extremists who will picket your company into crippling regulation after regulation until you can't even work anymore.

People are super against gene therapy because "we're trying to play God". It's sad, but sometimes the best defense against that, even if it's not very profitable (though it is 100% the best application for the therapy) is to hold up the "picketing our research is robbing these children of the right to live" emotional card. Even if that's what we really want to do, it still feels dirty. But then I remember that stem cell research was put on hold for years because crazy people were sending death threats to people and companies over it. Even when those stem cells were taken from the umbilical cords of kids born to happy parents.

If we started up with "look! It's just like plastic surgery!" the extremists will start throwing around buzzwords like "Nazis, Gattaca, Eugenics, racial extermination". You name it, I've even heard people start claiming that zombies were going to come of this, all because that's the plot of "I am Legend". Going slow by working on little kids with cancer is usually the morally best route, as well as the route that keeps you safest from the crazies.

[Cathal (Phone)]

I'd like to add that when people warn about the hazards in mammalian culture and gene therapy, it's not because we don't worry about haemophiliac kids. That's a bit unfair. I want to see cheap drugs for people who need them, too, and I think India is doing a great deed by challenging medical patents for that reason.

What I don't want is for people who care so much, like yourself, to end up harming yourselves or others in your rush to help. You're right to challenge the "precautionary principal" but if you sicken yourself or others trying to cure people of haemophilia DIY, you'll only make it harder for others to solve the problem. As a martyr for the precautionary extremists, you'll end up creating new and excessive laws, oversight and moratoriums.

And that's all beside the point that I would personally be very sad if you got hepatitis from your experiments.

On 9 April 2015 13:21:29 GMT+01:00, 'SC' via DIYbio <diy...@googlegroups.com> wrote:

Sunil,

 

Are you looking for guidance for your project, or do you just want to rant and insult the group members who have tried to help you?  I've made some suggestions which you have ignored. 

 

 

 

--
Sent from my Android device with K-9 Mail. Please excuse my brevity.

[Sunil Phani]

no,  I sincerely thank each and everyone in this group for passing their valuable suggestions and especially your suggestions were a real value addition 

and my intention is neither to rant or insult the group members but this continues rhetoric  is just making me feel loose all my hopes and some people are asking for citations and references for this noble cause I am not trying to build any business out of it I am just trying to help an NGO where you can see children crippled and with no future or hope in their eyes in this country we nither get social support from government nor get insurance cover for pre-existing diseases like hemophilia they are all leading pathetic life my efforts are just out of empathy for those kids, and tragically in this part of the world children are still dying cause of lack of required no of factors availability and this year we already lost 3 children aged less than 14  and that is making me soo stressed out too,

 we are all associated to a noble cause of this NGO to  see hemophiliacs without disability and children without pain, I have not ignored any of your suggestions and i want to reiterate once again that we are not going to try on any human beings we just want to explore theoretical possibilities via find appropriate solution and articulate it

and i will soon come out with my workout which I am carrying out with your suggestions i am really progressing with suggestions by humble people like you in this group  and i thank sincerely each and everyone from bottom of my heart 

Regards 

Sunil

On Thu, Apr 9, 2015 at 5:51 PM, 'SC' via DIYbio <diy...@googlegroups.com> wrote:

Sunil,

 

Are you looking for guidance for your project, or do you just want to rant and insult the group members who have tried to help you?  I've made some suggestions which you have ignored. 

 

 

 

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[SC]

Sunil,

 

You have a very noble motive.  The best way you can help the children with this disease is to take the project seriously.  A strong hope is not enough; you will need calculations and references, yes.  You should not jump into such a project without proper understanding and preparation.

 

When you are ready to continue your project in a serious way, feel free to post here and I will help you.  If you reject my suggestions and everyone else's, for example that mammalian cell culture is not recommended for beginners,  no one will be able to help you regardless of how sincere your motive is. 

[Sunil Phani]

Hey friends after serious work we finally annotated Plasmid vector with synthetic coagulation factor VIII gene in it, 

anyone has the facility to synthesize it for me, please

I will share the annotation in any format per requirement  

Thanks a Ton

On Saturday, January 17, 2015 at 7:31:14 AM UTC+5:30, Sunil Phani wrote:

[SC]

Hi Sunil,

Congratulations!

Did you use a bacterial plasmid?

[Sunil Phani]

i am using pHLsec as plasmid courtesy Oxford protein production lab.

let me sharing you the entire details about the pHL

Regards

Sunil Phani

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[Sunil Phani]

Hey Mega after serious work we finally annotated Plasmid vector with synthetic coagulation factor VIII gene in it, 

anyone has the facility to synthesize it for me, please

I will share the annotation in any format per requirement  

Thanks a Ton

Sunil Phani

On Mon, Jan 26, 2015 at 3:43 PM, Mega [Andreas Stuermer] <masters...@gmail.com> wrote:

While the pVIB plasmid is shipping, you might want to look into immunology a bit (though it is a very big and complex field), what would happen if you injected crudely purified protein into a human. What are the negative effects, and how are they induced.

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[Biotech Ryan]

Hi Sunil, have you thought of using arcturus.io (Arcturus Biocloud)? We're currently planning on making open source human insulin in E.Coli and the team there could help you with that (as well as setting up a team page to get advice).

I highly recommend checking them out and it'll be a lot cheaper and faster than setting up your own lab (they can mail you your construct and you can watch it online as the robot makes it).

Best,
Ryan

[Sunil Phani]

hey Rayan,  

we are amazed to learn about significant work you put in at arcturus.io Open Source Biotechnology for Everyone sound awesome and I am interested, 

your site can contribute a lot to our cause “Hemophilia without Disability and Children without pain”

as we are also here trying to work in crowdsourcing model only our goal is to 

produce recombinant Factor VIII we are willing to get engaged.

please tell me how to collaborate with you guys  and also interested to know how to setting up team page 

Thanks & Regards, Sunil Phani

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[Cathal Garvey]

Very topical:
http://www.economist.com/blogs/freeexchange/2015/07/greeces-economy-under-capital-controls

The EU is crushing Greece under boot-heel, and among the earliest
victims are Thyroxine and Insulin-dependent Greeks.

I wrote about the need for DIY Insulin/Thyroxine years ago and it's
still relevant; people underestimate how brittle the supply chains of
advanced medicine really are:
http://www.indiebiotech.com/?p=135

Given the difficulty of purifying blood-safe Insulin, Thyroxine (which
is orally bioavailable) might be a more immediately valuable project.
But with Sothic at IndieBio EU aiming to make Limulus Amoebocyte Lysate
(LAL) far, far more plentiful in the near future, perhaps LAL-testing of
stuff for safe injection will become more commonplace? I'm imagining a
market route something like:

1. DIY LAL test becomes available for intravenous drug users as part of
a harm-reduction strategy (GOOD IDEA)
2. Test gets re-appropriated to test batches of purified Factor VIII or
Insulin for human use, off-grid, by people who cannot access safe
medicine otherwise (Greece, much of India, for example).

Interesting times ahead.

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--
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Now running in Cork, Ireland May->July
Learn more at indie.bio and follow along!
Twitter: @onetruecathal
Phone: +353876363185
miniLock: JjmYYngs7akLZUjkvFkuYdsZ3PyPHSZRBKNm6qTYKZfAM
peerio.com: cathalgarvey

[Sunil Phani]

well said Cathal Garvey grate to be hearing your straight forward opinion from you,

i am really a grate admirer of your work as a matter of fact yesterday only i referred 

your article on this same topic in arcturus.io OSInsulin forum 

yes you are correct one has to prioritize research activities on vital issues first like

Factor VIII and Insulin/Thyroxine which are among the WHO recognized life saving drugs 

yet most of these drugs are propitiatory labeled only neither there is any generic 

formulation nor any major opensource project under progress. and these opensource 

projects too need to take up some bold innovative steps rather than thinking 

conservatively always like old school pharma companies we don't have any 

API's to protect 

hoping for democratization in its true essence 

[syntechbio]

Totally agree,

These are main issues that we cara about in arcturus.io , that is why we trying to give access to the tools that could help these projects. 

Sunil just opened this topic in our arcturus forum, lets also discuss there your plans to see how we can help you guys,

thanks, 

[Sunil Phani]

friends join us at https://forum.arcturus.io/t/open-source-coagulation-factor-viii/50 to discuss in developing Open Source Coagulation Factor VIII arcturus.io has came up with a cool platform with all developing tools in modular approach 

[Sunil Phani]

can i put forwarding few viewpoints just as thought provoking possibilities 

for discussing "why we always end up at bacterium as cell line always and
for every single recommended protein"

 

yes guys I accept it's less complex, less expensive and well established 
over decades and as every scientist I too believe in Occam's razor "law of parsimony", 

"when you have two competing theories that make exactly the same predictions, the simpler one is the better." 

but it Shouldn't stop us ideation on even simpler and everlasting approaches as Occam's razor also states 

"Everything should be made as simple as possible, but not simpler." why can't we adopt 

Agrobacterium-mediated Transient Protein Production in Nicotiana as it gives even 

simpler and everlasting approach to producing Protein of our interest, 

let me chalk down few Advantages and disadvantages of Agrobacterium.

 

Advantages:
- technically simple
- yields relatively uncomplicated insertion events (low copy number,minimal rearrangements)
- unlimited size of foreign DNA
- efficient (for most plants)
- adaptable to different cell types, culture procedures (protoplasts,tissue sections, “non-culture” methods)
- transformants are mitotically and meiotically stable

Disadvantages
- host range is limited: not all plants may be susceptible to Agrobacterium
- with susceptible plants, accessible culture/regeneration systems must be adaptable to Agrobacterium-mediated gene transfer

 

pls friends let me know your opinion in this regard

 

Regard 
Sunil Phani

[IgF1]

Hi, Sunil - Congratulations on your single-minded purpose, noble goals, success, and the grace to deal with what I can only describe as pathetic, insecure shits who feel like they can talk down to you (or in the third person) because you asked for their help with grace and humility, and because you didn't spell-check your responses! All the mumbo-jumbo about protocol and inability to replicate and money needed yada yada was such a display of counter-productive, hypocritical, mean-minded garbage that I laughed out loud when you handed them their butts on a plate.

Good for you, Sunil! And, congratulations and best wishes for all your future endeavors.